Fig. 3

SMN protein abundance is low in newborns, but SMN-VUS proteins are thermostable (A) Representative western blot and quantification of protein lysates from EBV-transformed lymphoblastoid cell lines were performed as previously described.24,25 SMN and actin (ACTB, loading control) levels are shown for P1 and P2, P1 family members, carriers, control, and individuals with SMA type IV. SMN/ACTB ratios are presented as mean ± SD (n = 4). Monoclonal antibodies against SMN (BD Biosciences) and ACTB (Proteintech) were used. The corresponding phenotype and SMN1/SMN2 genotype for each individual are provided in the table on the right. (B) C-terminal amino acid alignment of SMN across species, highlighting high conservation in the glycine zipper and oligomerization domain (red arrows) and lower conservation in the region containing the SMN-VUS. (C) AlphaFold3 structural predictions show that the SMN-VUS (orange) has a shorter helix and mildly altered tetramerization compared to SMN wild-type (WT) in green. (D) Thermostability assay of HA-tagged SMN1-WT, HA-SMN1-855VUS, and HA-SMN1-861VUS expression constructs, transfected in HeLa cells. Protein lysates were prepared after heating from 36°C to 51°C or kept on ice (control). Actin (ACTB), a well-known thermostable protein, was used as a reference. Western blot and immunostaining were performed using HA (Jackson ImmunoResearch), SMN (BD Biosciences), and ACTB (Proteintech) antibodies. (E) Quantifications of the Western blots included HA/control, HA/ACTB, HA/control/ACTB, and SMN/ACTB. SEM from four independent experiments showed no significant differences between SMN-WT and SMN-VUS proteins.